Development of NanoFludarabine for Pediatric Leukemia using LCP Nanotechnology NanoFludarabine is Qualiber's nanoparticle formulation of fludarabine phosphate (Fludara), a drug widely used for treating leukemia and other malignancies. While fludarabine phosphate is effective against tumors, it also has significant toxicities. Many of these side effects can potentially be minimized or avoided by modifying the biodistribution of fludarabine phosphate. Furthermore, fludarabine phosphate's therapeutic index can be improved if we can preferentially deliver the drug to cancer cells using nanoparticles which have been targeted to cancer cells. Our innovation is the encapsulation of fludarabine phosphate in a novel nanoparticle formulation, called Lipid-Calcium-Phosphate (LCP). The new formulation of fludarabine phosphate in LCP nanoparticles is named NanoFludarabine. The calcium phosphate core of LCP would encapsulate fludarabine phosphate with a high efficiency and rapidly dissolve in the acidic endosome releasing fludarabine phosphate in the cytoplasm where it is further phosphorylated to fludarabine triphosphate and transported to the nucleus for inhibition of DNA synthesis. In this Phase I, Qualiber will formulate LCP for the delivery of fludarabine phosphate (NanoFludarabine) and characterize them for in vitro activity and determine feasibility for development as pediatric leukemia treatment. Aim 1. Increase Current Production Scale of Encapsulated Fludarabine Phosphate in LCP Nanoparticles by 10 Fold with In-Process Controls (IPC) Qualiber aims to test the feasibility of scalable process to encapsulate fludarabine phosphate with solid analytical measurements. The preliminary results show that fludarabine phosphate can be encapsulated into LCP particles at lab scale with batch size of 1 mg of fludarabine phosphate encapsulated. Two challenges exist at this stage: (i) small scale of production and (ii) lack of process controls Aggregation of particles was observed partly due to a low surface zeta potential. Successful production to improve the scale of nanoparticles to 10x of current scale with batch size of >10 mg of fludarabine phosphate will be the criteria for progressing to Aim 2. Also, in-process controls such as drug encapsulation efficiency (>40%), particle size and poly-dispersity will be put in place to ensure future process designs for a scalable manufacturing process that would allow further pre-clinical development and clinical studies regulated by FDA. Aim 2. Select One Targeted Ligand and Make NanoFludarabine to Target Leukemia with Proof of Concept (POC) In Vitro Studies NanoFludarabine nanoparticles produced in the above scale-up process will be further targeted to leukemia cells. Qualiber is planning to make NanoFludarabine with at least two different ligands to target (i) CD44 receptor with Hyaluronan/Hyaluronic Acid (HA) and (ii) Folate receptor with PEGylated folic Acid (FA). Free fludarabine phosphate, NanoFludarabine without ligand, NanoFludarabine with HA or FA targeting ligands will be tested in in vitro cell culture models. The results will be used to determine the following: (a) Is NanoFludarabine more efficacious than free fludarabine phosphate and (b) Which targeting ligand provides highest cell uptake via endocytosis of the nanoparticles. One ligand will be chosen based on the targeting efficacy, availability and manufacturing feasibility to move forward. Successfully demonstrating that NanoFludarabine (with HA or FA targeting ligand) has a IC50 values at least 2-5 fold lower than free fludarabine phosphate will be a key milestone for progressing the product for further preclinical development and Phase II SBIR grant.